Systems and methods for distinguishing stimulated emissions as a means of increasing the signal of fluorescence microscopy

ABSTRACT

Embodiments of a fluorescence microscopy system that employs a technique for distinguishing stimulated emission as a means for enhancing signal strength of fluorescent markers are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a non-provisional application that claims benefit to U.S.provisional patent application Ser. No. 62/072,218, filed on Oct. 29,2014, which is incorporated by reference in its entirety.

FIELD

The present document generally relates to fluorescence microscopy and inparticular to systems and methods of distinguishing stimulated emissionsas a means of increasing the signal from fluorophores.

BACKGROUND

Fluorescence microscopy is an invaluable tool for biologists, whichprovides high-resolution, high-speed, protein-specific imaging in livingcells, tissues, and animals. In particular, fluorescence microscopy usesmarkers which absorb light and spontaneously re-emit that light at adifferent wavelength. The nanosecond-scale lag between absorption andspontaneous emission limits how much light a fluorescent marker emitsper second, which fundamentally limits measurement speed and precision.For example, seeing individual steps of a RNA polymerase as it movesalong a DNA strand requires a fluorescent marker which emits thousandsof photons per millisecond, much more than typical fluorescent proteinscan produce. As such, greatly increasing the brightness andphotostability of fluorescent markers would enable high speed, highprecision measurements which are currently impossible.

Spontaneous emission is not the only way for an excited marker to emitlight after absorption. If an excited marker is illuminated with lightof the proper color, it can also be “stimulated” to emit. Since the rateof stimulated emission can be much faster than spontaneous emission,stimulated emission can be several orders of magnitude brighter thanspontaneous emission, an exciting possibility for improving fluorescentmarker brightness. However, stimulated emission is difficult todistinguish from the stimulating light—it is the same color, the samephase, the same polarization, and, in bulk materials, goes the samedirection. Noise and background from the stimulating beam is thereforedifficult to reject, which negates the advantage of stimulated emissionfor increasing marker brightness. As such, no method to cleanlydistinguish stimulated emission from the stimulating beam is currentlyknown.

SUMMARY

In one embodiment, a fluorescence microscopy system includes anexcitation source for generating an excitation light beam along a firstaxis and a stimulation source for generating a stimulation light beamalong a second axis that is in perpendicular orientation relative to thefirst axis, wherein the excitation light beam and the stimulation lightbeam have different wavelengths. In addition, a sample having one ormore fluorescent markers is provided which may be illuminated by theexcitation light beam such that the one or more fluorescent markersassume an excited state and then illuminated again by the stimulationlight beam to generate a stimulated emission by the initially excitedsample. An objective lens is oriented along the second axis forcapturing and focusing the stimulation light beam and stimulatedemission emitted by the one or more fluorescent markers. A stimulationlight beam block component is positioned at the focal point of theobjective lens such that the stimulation light beam is blocked whileallowing the stimulated emission to pass. Finally, a detector isprovided for detecting the stimulated emission.

In another embodiment, a method for fluorescence microscopy includesilluminating one or more fluorescent markers in a sample with anexcitation light beam from an excitation source oriented along a firstaxis such that the one or more fluorescent marker assume an excitedstate; illuminating the one or more fluorescent markers in the excitedstate with a stimulation light beam from a stimulation source orientedalong a second axis that is in perpendicular relation to the first axis,wherein a stimulated emission is generated by the one or morefluorescent markers when illuminated by the stimulation light beam, andwherein the stimulated light beam and the excitation light beam havedifferent wavelengths; focusing the stimulation light beam and thestimulated emission through an objective lens; positioning a stimulatedlight beam block component along the second axis such that thestimulated light beam is focused onto and blocked by the stimulatedlight beam block component, and wherein the stimulated emission is notblocked by the positioning of the stimulated light beam block; anddetecting the stimulated emission by a detector.

In yet another embodiment, a fluorescence microscopy system includes anexcitation source for generating an excitation light beam along a firstaxis and a stimulation source for generating a stimulation light beamalong a second axis that is in perpendicular relation relative to thefirst axis, wherein the excitation light beam and the stimulation lightbeam have different wavelengths. A combined excitation/stimulation lightbeam is generated by the intersection of the excitation light beam withthe stimulation light beam with an excitation objective lens orientedalong the first axis for capturing the combined excitation/stimulationlight beam. A sample having one or more fluorescent markers is providedin the focal plane of the excitation objective lens, wherein the one ormore fluorescent markers are illuminated by the combinedexcitation/stimulation light beam for producing a stimulated emission.In addition, a detection objective lens is oriented in perpendicularorientation relative to the excitation objective for capturing andfocusing the stimulated emission emitted by the one or more fluorescentmarkers of the sample and a detector for detecting the stimulatedemission from the detection objective lens.

In a further embodiment, a method for fluorescence microscopy includesgenerating an excitation light beam along a first axis; generating astimulation light beam along a second axis that is in perpendicularorientation relative to the first axis, wherein the excitation lightbeam and the stimulation light beam have different wavelengths;intersecting the excitation light beam with the stimulation light beamsuch that a combined excitation/stimulation light beam is generated anddirected along the second axis focusing the combinedexcitation/stimulation light beam through an excitation objectiveoriented along the second axis onto a sample having one or morefluorescent markers such that the one or more fluorescent markers areilluminated by the combined excitation/stimulation light beam and emit astimulated emission; focusing the stimulated emission through adetection objective lens oriented along a third axis that is inperpendicular relation relative to the second axis and in parallelrelation relative to the first axis; and detecting the stimulatedemission with a detector.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a simplified illustration of one embodiment of a fluorescencemicroscopy system; and

FIG. 2 is a simplified illustration of another embodiment of thefluorescence microscopy system.

Corresponding reference characters indicate corresponding elements amongthe view of the drawings. The headings used in the figures do not limitthe scope of the claims.

DETAILED DESCRIPTION

Various embodiments of a fluorescence microscopy system that employs atechnique for distinguishing stimulated emission as a means of enhancingsignal strength of fluorescent markers for detection are disclosed.Referring to the drawings, embodiments of a fluorescence microscopysystem are illustrated and generally indicated as 100 and 200 in FIGS. 1and 2.

As shown in FIG. 1, one embodiment of a fluorescent microscopy system100 that employs one technique for distinguishing stimulated emission asa means of enhancing signal strength of fluorescent markers is shown. Inone arrangement, an excitation beam 106 is used to illuminate a sample102 along an axis 302 in which one or more fluorescent molecules 110(e.g. fluorescent markers) associated with the sample 102 are excited bythe excitation light beam 106. For example, the excitation light beam106 may be generated by an excitation source 101, such as a laser, thatemits a single light beam having a wavelength of about 488 nm.

Once the fluorescent molecules 110 assume an excited state afterillumination by the excitation light beam 106, a stimulation light beam104 is then used to illuminate the sample 102 along an axis 300 that isin perpendicular relation to the axis 302 of the excitation light beam106. In some embodiments, the stimulation light beam 104 may begenerated by a stimulation source 103, such as a laser, in which thestimulation light beam 104 comprises collimated light having awavelength of about 550 nm or any other wavelength that is differentthan the wavelength of the excitation light beam 106. In someembodiments, the stimulation light beam 104 may be focused onto thesample 102 using an optics arrangement (not shown) to illuminate theexcited fluorescent molecules 110 immediately after initial excitationoccurs in the sample 102 by the excitation light beam 106. It wasdiscovered that if the excited fluorescent molecules 110 are illuminatedonce again with light of the proper wavelength (e.g., stimulation lightbeam 104) after initial excitation, the excited fluorescent molecules110 will be stimulated to emit a stimulated emission 108 that can bedetected. In particular, after initial excitation of the fluorescentmolecules 110 by the excitation light beam 106, the fluorescentmolecules 110 emit light in different wavelengths. Once the fluorescentmolecules 110 are initially excited, the fluorescent molecules 110 maythen be illuminated again by the stimulation light beam 104 having awavelength that the excited fluorescent molecule 110 may have emittedduring initial excitation in order to reinforce light of that specificwavelength by the stimulated emission 108. However, the stimulatedemission 108 generated by each fluorescent molecule 110 is difficult todistinguish from the stimulated light beam 104 since both travel alongthe same axis 300.

In some embodiments, the fluorescent molecules 110 may be fluorescentmarkers that are distributed in a sparse or non-uniform manner along thesample 102.

To distinguish and separate the stimulation light beam 104 from thestimulated emission 108, the fluorescence microscopy system 100 includesa stimulation light beam block component 112 associated with anobjective lens 114 for effectively blocking the stimulation light beam104 from detection by a detector 118 along axis 300. For example, thestimulation light beam block component 112 is positioned at the focalpoint of the objective lens 114 such that stimulation light beam 104 isfocused onto the stimulation light beam block component 112 and blockedfrom detection by the detector 118, while allowing the stimulatedemission 108 to be imaged around the stimulation light beam blockcomponent 112 and focused by a lens 116 onto the detector 118. In thisarrangement, the fluorescence microscopy system 100 allows thestimulated emission 108 to be generated sooner by the fluorescentmolecule 110 with the stimulated emission 108 having a particulardesired wavelength.

In another embodiment, the fluorescence microscopy system, designated200, also employs a technique for distinguishing stimulated emission asa means of enhancing signal strength of fluorescent markers is shown. Inone arrangement, an excitation light beam 202 is generated by anexcitation source 201 similar to the excitation source 101, whichgenerates the excitation light beam 202 directed along a first axis 400and is combined with a stimulation light beam 204 generated by astimulation source 203 similar to stimulation source 103, which directsthe stimulated light beam 204 along a second axis 402 that is inperpendicular relation to the first axis 400 to generate a combinedexcitation/stimulation light beam 205 at an intersection point 215(represented in FIG. 2 as a circle). The combined excitation/stimulationlight beam 205 generated by the intersection of the excitation lightbeam 202 with the stimulation light beam 204 at a perpendicular angleilluminates a sample 208 along second axis 402. In particular, thecombined excitation/stimulation light beam 205 is captured by anexcitation objective 216 that focuses the combinedexcitation/stimulation light beam 205 along the second axis 402 toilluminate the sample 208 in which one or more fluorescent molecules 210(e.g. fluorescent markers) associated with the sample 208 are excited bythe combined excitation/stimulation light beam 205. Thisexcitation/stimulation of the sample 208 generates a stimulated emission206 by the one or more fluorescent molecules 210 associated with thesample 208.

Once the fluorescent molecules 210 are excited by the combinedexcitation/stimulation light beam 205, the combinedexcitation/stimulation light beam 205 continues along second axis 402 asshown in FIG. 2. A portion of the stimulated emission 206 is emitted inthe direction of a third axis 404, which is in parallel orientationrelative to the first axis 400 and in perpendicular orientation relativeto the second axis 402, is captured by a detection objective lens 218.In one arrangement, the excitation objective lens 216 is inperpendicular orientation relative to the detection objective lens 218,which allows the stimulated emission 206 directed along third axis 404to separate from the combined excitation/stimulation light beam 205directed along second axis 402. As such, the fluorescence microscopysystem 200 does not require the excitation objective lens 216 to includea stimulation light beam block component 112 of the fluorescencemicroscopy system 200 in order to separate the combinedexcitation/stimulation light beam 205 from the stimulated emission 206.

Once the stimulated emission 206 is captured by the detection objectivelens 218, the stimulated emission 206 is focused onto a detector 210 forcapturing images of the stimulated emission 206 emitted by the sample208. In some embodiments, a filter 214 may be interposed between thedetection objective 218 and the detector 210 in order to filter outportions of the stimulated emission 206.

It should be understood from the foregoing that, while particularembodiments have been illustrated and described, various modificationscan be made thereto without departing from the spirit and scope of theinvention as will be apparent to those skilled in the art. Such changesand modifications are within the scope and teachings of this inventionas defined in the claims appended hereto.

What is claimed is:
 1. A fluorescence microscopy system comprising: anexcitation source for generating an excitation light beam along a firstaxis; a stimulation source for generating a stimulation light beam alonga second axis that is in perpendicular orientation relative to the firstaxis, wherein the excitation light beam and the stimulation light beamhave different wavelengths; a sample having one or more fluorescentmarkers which are illuminated by the excitation light beam such that theone or more fluorescent markers assumes an excited state and thenilluminated again by the stimulation light beam to generate a stimulatedemission by the initially excited sample; an objective lens orientedalong the second axis for capturing and focusing the stimulation lightbeam and stimulated emission emitted by the one or more fluorescentmarkers; a stimulation light beam block component positioned at thefocal point of the objective lens such that the stimulation light beamis blocked while allowing the stimulated emission to pass; and adetector for detecting the stimulated emission.
 2. The fluorescencemicroscopy system of claim 1, wherein the excitation light beam alongthe first axis is not captured by the objective lens.
 3. Thefluorescence microscopy system of claim 1, wherein the stimulation lightbeam is collimated.
 4. The fluorescence microscopy system of claim 1,further comprising: a lens in association with the objective lens forfocusing the stimulated emission onto the detector.
 5. The fluorescencemicroscopy system of claim 1, wherein the stimulated emission is imagedaround the stimulation beam block component for detection by thedetector.
 6. The fluorescence microscopy system of claim 1, wherein theone or more fluorescence markers are distributed sparsely or in anon-uniform manner along the sample.
 7. The fluorescence microscopysystem of claim 1, wherein the wavelength of the stimulation emission isthe same wavelength that the one or more fluorescent markers can emitduring excitation of the sample by the excitation light beam.
 8. Thefluorescence microscopy system of claim 1, wherein the stimulation lightbeam has a wavelength of about 550 nm.
 9. The fluorescence microscopysystem of claim 1, wherein the stimulation source and the excitationsource comprise a laser.
 10. The fluorescence microscopy system of claim1, wherein the excitation light beam has a wavelength of about 488 nm.11. The fluorescence microscopy system of claim 1, wherein thestimulation light beam block component is aligned along the second axisand positioned to block the stimulated light beam from the objectivelens.
 12. A method for fluorescence microscopy comprising: illuminatingone or more fluorescent markers in a sample with an excitation lightbeam from an excitation source oriented along a first axis such that theone or more fluorescent marker assume an excited state; illuminating theone or more fluorescent markers in the excited state with a stimulationlight beam from a stimulation source oriented along a second axis thatis in perpendicular relation to the first axis, wherein a stimulatedemission is generated by the one or more fluorescent markers whenilluminated by the stimulation light beam, wherein the stimulated lightbeam and the excitation light beam have different wavelengths; focusingthe stimulation light beam and the stimulated emission through anobjective lens; positioning a stimulated light beam block componentalong the second axis such that the stimulated light beam is focusedonto and blocked by the stimulated light beam block component, andwherein the stimulated emission is not blocked by the positioning of thestimulated light beam block; and detecting the stimulated emission by adetector.
 13. The method of claim 12, wherein the stimulated light beamblock is positioned at the focal point of the objective lens andoriented along the second axis.
 14. The method of claim 12, furthercomprising: focusing the stimulated emission using a lens onto thedetector.
 15. A fluorescence microscopy system comprising: an excitationsource for generating an excitation light beam along a first axis; astimulation source for generating a stimulation light beam along asecond axis that is in perpendicular relation relative to the firstaxis, wherein the excitation light beam and the stimulation light beamhave different wavelengths; a combined excitation/stimulation light beamthat is generated by the intersection of the excitation light beam withthe stimulation light beam; an excitation objective lens oriented alongthe first axis for capturing the combined excitation/stimulation lightbeam; a sample having one or more fluorescent markers in the focal planeof the excitation objective lens, wherein the one or more fluorescentmarkers are illuminated by the combined excitation/stimulation lightbeam for producing an stimulated emission; a detection objective lensoriented in perpendicular orientation relative to the excitationobjective for capturing and focusing the stimulated emission emitted bythe one or more fluorescent markers of the sample; and a detector fordetecting the stimulated emission from the detection objective lens. 16.The fluorescence microscopy system of claim 15, further comprising: afilter in association with the detection objective lens for filteringthe stimulated emission focused by the detection objective lens.
 17. Thefluorescence microscopy system of claim 15, wherein the wavelength ofthe stimulation emission is a wavelength that the one or morefluorescent markers can emit during excitation by the excitation lightbeam.